Design, synthesis, and antitumor activity of PLGA nanoparticles incorporating a discovered benzimidazole derivative as EZH2 inhibitor

Chemico-Biological Interactions 344 (2021) 109530

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Chemico-Biological Interactions

journal homepage: www.elsevier.com/locate/chembioint

Research paper

Design, synthesis, and antitumor activity of PLGA nanoparticles

incorporating a discovered benzimidazole derivative as EZH2 inhibitor

,

Hoda A. Elkot^a, Ibrahim Ragab^a, Noha M. Saleh^{b *}, Mohamed N. Amin^c, Sara T. Al-Rashood^d,

,

**

Shahenda M. El-Messery^e, Ghada S. Hassan^f

^aFaculty of Pharmacy, Mansoura University, P.O. Box 35516, Mansoura, Egypt

^bDepartment of Pharmaceutics, Faculty of Pharmacy, Mansoura University, P.O. Box 35516, Mansoura, Egypt

^cDepartment of Biochemistry, Faculty of Pharmacy, Mansoura University, P.O. Box 35516, Mansoura, Egypt

^dDepartment of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh, 11451, Saudi Arabia

^eDepartment of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Mansoura University, P.O. Box 35516, Mansoura, Egypt

^fDepartment of Medicinal Chemistry, Faculty of Pharmacy, Mansoura University, P.O. Box 35516, Mansoura, Egypt

A R T I C L E I N F O

A B S T R A C T

Keywords:

Synthesis

Purpose: Targeting enhancer of zeste homolog 2 (EZH2) can represent a hopeful strategy for oncotherapy. Also,

the use of PLGA-based nanoparticles as a novel and rate-controlling carrier system was of our concern.

Methods: Benzimidazole derivatives were synthesized, and their structures were clariﬁed. In vitro antitumor

activity was evaluated. Then, a modeling study was performed to investigate the ability of the most active

compounds to recognize EZH2 active sites. Compound 30 (Drug) was selected to conduct pre-formulation studies

and then it was incorporated into polymeric PLGA nanoparticles (NPs). NPs were then fully characterized to

select an optimized formula (NP₄) that subjected to further evaluation regarding antitumor activity and protein

expression levels of EZH2 and EpCAM.

EZH2 inhibitor

PLGA

Nanoparticles

Antitumor activity

EpCAM

Results: The results showed the antitumor activity of some synthesized derivatives. Docking outcomes demon-

strated that Compound 30 was able to identify EZH2 active sites. NP₄exhibited promising ﬁndings and proved to

keep the antitumor activity of Compound 30. HEPG-2 was the most sensitive for both Drug and NP₄. Protein

analysis indicated that Drug and NP₄had targeted EZH2 and the downstream signaling pathway leading to the

decline of EpCAM expression.

Conclusions: Targeting EZH2 by Compound 30 has potential use in the treatment of cancer especially hepato-

cellular carcinoma.

trimethylation of lysine 27 in histone H3 (H3K27me3). The trimethy-

lation process results in a repressive chromatin environment. Therefore,

PRC2 has a vital role in silencing gene expression. Enhancer of zeste

homolog 2 (EZH2) is a histone-lysine N-methyl transferase enzyme that

1. Introduction

Epigenetic mechanisms are vital for the ordinary growth and main-

tenance of different gene expression patterns. The failure to properly

maintain such heritable epigenetic characters can cause undesirable

activation or inhibition of various gene expressions which in turn end

with the induction of several diseases. It has been realized that epige-

netic abnormalities are involved in the initiation and progression of

cancer [1]. Among various pathways, epigenetic changes such as histone

modiﬁcations by covalent or noncovalent mechanisms and DNA

methylation have been veriﬁed as promising approaches for oncother-

apy [2]. Polycomb repressive complex 2 (PRC2) is accountable for di- or

represents a subunit of PRC2 [3]. Moreover, EZH2 can function inde-

pendently of the other PRC2 subunits, methylating non-histone proteins

[4]. EZH2 over activation is linked to overexpression of epithelial cell

adhesion molecule (EpCAM) expressing cancer cells. EpCAM + cells are

usually referred to as cancer stem cells, which contribute to tumor

malignancy and resistance to chemo/radiotherapy [5]. An association

has been well documented between overexpression of EZH2 and

aggressiveness of breast cancer, prostate cancer, and especially hepa-

tocellular carcinoma (HCC) [6]. EZH2 affects the expression of very

* Corresponding author.

** Corresponding author.

E-mail address: nunu_ramy@yahoo.com (N.M. Saleh).

https://doi.org/10.1016/j.cbi.2021.109530

Received 22 December 2020; Received in revised form 16 May 2021; Accepted 19 May 2021

Available online 23 May 2021

H.A. Elkot et al.

Chemico-Biological Interactions 344 (2021) 109530

Abbreviations

MCF-7

MTT

Breast cancer

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium

Drug

DIW

DLS

Compound 30

bromide

Deionized water

NPs

Polymeric nanoparticles

Dynamic Light Scattering

Differential Scanning Calorimetry

Encapsulation efﬁciency

PB_7.4/MeOH₂₀Phosphate buffer pH 7.4 containing 20% v/v

methanol

DSC

EE%

PBS

Phosphate buffer saline

Prostate cancer

EpCAM Epithelial cell adhesion molecule

PC-3

PDI

EPR

Enhanced permeation and retention effect

Enhancer of zeste homolog 2

Polydispersity index

Poly(D, ^L-lactide-co-glycolide)

Polycomb repressive complex 2

Polyvinyl alcohol

EZH2

FBS

PLGA

PRC2

PVA

TEM

T_M

Fetal Bovine serum

FT-IR

Fourier Transform Infrared Spectroscopy

H3K27me3 Trimethylation of lysine 27 in histone H3

HEPG-2 Hepatocellular carcinoma

Transmission electron microscopy

Melting points

HLB

K_o/w

LC%

Hydrophilic lipophilic balance

Octanol-water partition coefﬁcient

Loading capacity

Y%

Percent yield

Z-average Average size

ζ

Zeta potential

important tumor suppressor genes such as SNF5 leading to its suppres-

sion. Hence, EZH2 is considered a talented therapeutic target for the

treatment of hypermethylation-related cancers [7].

concealed with a leaky vasculature that drives leakage of NPs from the

vasculature through these permeable endothelial tissues to preferen-

tially accumulate in the solid tumors. This phenomenon is called

enhanced permeation and retention effect (EPR) and it is useful to

promote passive targeting of the chemotherapeutic agent to tumor cells

[14]. Hence, tumor tissue by EPR is more permeable owing to the wide

fenestrations of the angiogenic vasculature. Besides, NPs are more

retained in tumor regions due to a lack of lymphatic around them. Such a

situation reducing the exposure of healthy cells to chemotherapeutic

drugs [15].

Benzimidazole is a central heterocyclic compound and one of the

power structures found in a large number of natural products [8]. The

heterocyclic nucleus of benzimidazole is considered a “Master Key” in

medicinal chemistry. For FDA-approved pharmaceutical drugs, this

nucleus is found to be among the top ﬁve most common ﬁve-membered

aromatic nitrogen heterocycles. Several benzimidazole derivatives

bearing various substitutions at the C-2 position are widely distributed

in nature and possess versatile biological activities. By extensive bio-

activities such as anti-protozoal, anti-microbial, antioxidant, analgesic,

anti-inﬂammatory, anti-hypertensive, and anticancer activity, benz-

imidazole derivatives have an essential role in drug discovery. Benz-

imidazoles are considered important pharmacophores of cancer

research due to wide anticancer capacities with multiple mechanisms.

Their anticancer selectivity is contingent on the substitution of the

benzimidazole nucleus to provide a track for the development of novel

highly effective and target-speciﬁc benzimidazole-based anticancer

drugs. Hence, benzimidazole derivatives have attracted our attention in

this study and we had planned to synthesize a series of new benzimid-

azole derivatives [9]. The synthesized molecules with promising activ-

ities were planned to be incorporated into proper drug delivery systems.

Classical drug delivery involves frequent dosing, which usually results in

that a major part of the drug tends to be released rapidly after the

administration. Such a situation could cause oscillations of drug levels

between the side effects level and the minimum effective level with

alternating periods of toxicity and ineffectiveness [10]. Hence,

controlled drug delivery could be suggested as a strategy to overcome

the drawbacks of the conventional one. Controlling the release of drugs

can be accomplished by combining appropriate carrier materials with

active agents to improve the pattern of drug release, the systemic cir-

culation time, as well as the site drug delivery i.e. pharmacokinetics and

pharmacodynamics [11].

Speciﬁc attention has been lately focused on polymeric NPs prepared

of several smart biodegradable and biocompatible polymers such as Poly

(D, ^L-lactide-co-glycolide) (PLGA). PLGA-based NPs of many drugs are

currently holding FDA approval for several therapeutic applications

[16]. PLGA is broadly used to sustain the release of many drugs, enhance

the in vivo oral bioavailability and therapeutic efﬁcacy [17]. Addition-

ally, PLGA can protect drugs from early degradation and hepatic pass

metabolism. The release pattern and rate of a drug could be planned by

augmenting particle size, surface charge, morphology, and loading ef-

ﬁciency of PLGA-based NPs [18].

In light of the above details, the present study focused on discovering

a benzimidazole-based epigenetic regulator as antagonists of EZH2.

Also, their antitumor activity and selectivity were aimed to be explored.

Besides, we aimed to enhance the physicochemical properties of these

EZH2 inhibitors via incorporation in novel nanocarrier systems based on

PLGA NPs. Keeping antitumor activity after incorporation of an EZH2

inhibitor in NPs was of our concern.

2. Experimental

2.1. Materials

PLGA (Purasorb PDLGA®5010, MWt 153 KDa, Purac Biomaterials,

Holland), Tween 20 (Polyoxyethylene(20) sorbitan monolaurate, LOBA

Chemie Pvt. Ltd., India), Polyvinyl alcohol (PVA, MWt 14 KDa, BDH,

USA), RPMI-1640 medium and DMEM-high glucose medium (Lonza,

UK), MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bro-

mide] (Serva, Heidelberg, Germany), dimethylsulfoxide (DMSO, Sigma-

Aldrich, St. Louis, USA), Fetal Bovine Serum (FBS, Gibco, UK), Methanol

and Acetone (El-Nasr Pharmaceutical Chemicals Co., ADWIC, Cairo,

Egypt). EZH2 protein ELISA kit (ab264632, Abcam, Cambridge, UK),

EpCAM protein ELISA kit (LS-F7278, LSBio, Seattle, WA). All ﬁne re-

agents and chemicals were obtained from Aldrich Chemicals Co., USA.

Currently, scientists have explored the ﬁeld of nanotechnology to

develop a proper drug carrier system for the effective and controlled

delivery of loaded drugs to cancer cells. Due to their extraordinary

physicochemical properties, nanomaterials such as nanoparticles;

nanocapsules or nanospheres, nanoﬁbers, nanotubes, and nanovesicles

realize successful carrier matrices of many drugs [10]. Polymeric

nanoparticles (NPs) can be applied to deliver a drug in a rate-controlled

and occasionally targeted manner [12]. NPs can permeate through

biological barriers due to their reduced size associated with high surface

area [13]. The biodistribution of NPs enriches their cellular uptake and

improves the drug concentration in tissues. Regularly solid tumors are

2

H.A. Elkot et al.

Chemico-Biological Interactions 344 (2021) 109530

2.2. Synthesis of new benzimidazole derivatives

N, 12.02; S, 13.75. Found: C, 41.35; H, 2.71; N, 12.89; S, 14.17. MS m/z

(%): 232 (11.8, M⁺).

New benzimidazole compounds were synthesized, their melting

points (T_M, ^◦C) were determined (Mettler FP 80 melting point apparatus,

Mettler, Manchester, UK) and uncorrected. C, H, and N of all compounds

were analyzed, and they were found to agree with the suggested struc-

tures within ±0.4% of the theoretical values. ¹H, ¹³C NMR of the syn-

thesized compounds were recorded (JOEL 500 MHz FT spectrometer,

Japan) and chemical shifts were expressed in δ ppm concerning TMS. On

pre-coated silica gel (0.25 mm) GF₂₅₄plates (Merck, Germany), thin-

layer chromatography was performed, and the compounds were detec-

ted under a UV lamp at 254 nm. Silica gel with 60–230 mesh was applied

for routine separations of column chromatography.

2.2.3. Synthesis of 2-hydrazinyl-5,6-(un) substituted-1H-benzo[d]

imidazoles (10–12)

A solution of methylthio-benzoimidazole derivatives 7-9 (0.01 mol)

in 10 ml hydrazine hydrate was reﬂuxed for 12 h. The resultant solution

was then poured into iced water, extracted with methylene chloride, and

ﬁnally crystallized from ethanol to produce the desired products in good

yields.

2.2.3.1. 2-Hydrazinyl-1H-benzo[d]imidazole (10). Yield 75.4%, ¹H N

MR (500 MHz, DMSO‑d₆) δ 4.41 (s, 2H, NHNH₂), 6.38 (d, 2H, J = 8.0 Hz,

Ar–H), 7.10 (t, 2H, J = 7.3 Hz, Ar–H), 7.75 (s, 1H, NHNH₂), 10.22 (s, 1H,

NH). Elemental Analysis: Calcd: C, 56.74; H, 5.44; N, 37.81. Found: C,

56.91; H, 5.72; N, 38.16. MS m/z (%): 148 (3.1, M⁺).

2.2.1. Synthesis of 5,6-(un) substituted-1H-benzo[d]imidazole-2-thiols

(4–6)

An alkaline solution of a mixture of diaminobenzene derivative (1-3)

(0.01 mol) and carbon disulﬁde (2 ml), potassium hydroxide (0.4 g,

0.01 mol) in (30 ml) methanol has been prepared, reﬂuxed overnight.

The obtained solution was evaporated under reduced pressure, and the

residue obtained was washed with water, extracted with chloroform.

Then, the organic layer was separated and dried over anhydrous sodium

sulfate to obtain the required compounds.

2.2.3.2. 2-Hydrazinyl-5,6-dimethyl-1H-benzo[d]imidazole (11). Yield 6

7.2%, ¹H NMR (500 MHz, DMSO‑d₆) δ 1.73 (s, 6H, CH₃), 4.52 (s, 2H,

NHNH₂), 7.34 (s, 2H, Ar–H), 8.14 (s, 1H, NHNH₂), 10.42 (s, 1H, NH).

Elemental Analysis: Calcd: C, 61.34; H, 6.86; N, 31.79. Found: C, 61.92;

H, 7.08; N, 32.10. MS m/z (%): 176 (9.7, M⁺).

2.2.3.3. 5,6-Dichloro-2-hydrazinyl-1H-benzo[d]imidazole (12). Yield 8

0.6%, ¹H NMR (500 MHz, DMSO‑d₆) δ 4.67 (s, 2H, NHNH₂), 7.94 (s, 2H,

Ar–H), 9.37 (s, 1H, NHNH₂), 10.44 (s,1H, NH). Elemental Analysis:

Calcd: C, 38.74; H, 2.79; N, 25.81. Found: C, 39.00; H, 2.94; N, 25.49.

MS m/z (%): 216 (12.4, M⁺).

1

2.2.1.1. 1H-benzo[d]imidazole-2-thiol (4). Yield 85.6%, H NMR (500

MHz, DMSO‑d₆) δ 7.09 (d, 2H, J = 8.0 Hz, Ar–H), 7.21 (t, 2H, J = 8.5 Hz,

Ar–H), 9.32 (s, 1H, NH), 10.39 (s, 1H, SH). Elemental Analysis: Calcd: C,

55.98; H, 4.03; N, 18.65; S, 21.35. Found: C, 56.15; H, 3.99; N, 18.79; S,

21.66. MS m/z (%): 150.0 (15.4, M⁺).

2.2.4. Synthesis of (Z)-5,6-(un) substituted-2-(2-substituted benzylidene)

hydrazinyl)-1H-benzo[d]imidazoles (17–28)

2.2.1.2. 5,6-dimethyl-1H-benzo[d]imidazole-2-thiol (5). Yield 88.5%, ¹H

NMR (500 MHz, DMSO‑d₆) δ 1.52 (s, 6H, CH₃), 7.51 (s, 2H, Ar–H), 9.61

(s, 1H, NH), 11.61 (s, 1H, SH). Elemental Analysis: Calcd: C, 60.64; H,

5.65; N, 15.72; S, 17.99. Found: C, 60.74; H, 5.48; N, 16.10; S, 17.53. MS

m/z (%): 178.2 (12.3, M⁺).

A mixture of hydrazinyl derivatives 10-12 (0.01 mol) and the

appropriately substituted benzaldehyde (0.01 mol) in 30 ml ethanol

with a catalytic amount of acetic acid (1 ml) was reﬂuxed for 8 h. Then,

the solvent allowed to evaporate under reduced pressure, and the left

residue was crystallized by aqueous ethanol to get the required

compounds.

2.2.1.3. 5,6-dichloro-1H-benzo[d]imidazole-2-thiol (6). Yield 87.3%, ¹H

NMR (500 MHz, DMSO‑d₆) δ 7.94 (s, 2H, Ar–H), 9.51 (s, 1H, NH), 10.99

(s, 1H, SH). Elemental Analysis: Calcd: C, 38.38; H, 1.84; N, 12.79; S,

14.63. Found: C, 38.61; H, 1.72; N, 12.94; S, 15.08. MS m/z (%): 218.0

(21.3, M⁺).

2.2.4.1. (Z)-2-(2-(4-methoxybenzylidene) hydrazinyl)-1H-benzo[d]imida

1

zole (17). Yield 61.7%, H NMR (500 MHz, DMSO‑d₆) δ 3.75 (s, 3H,

–

OCH ), 6.47 (s, 1H, N CH), 7.32 (d, 2H, J = 8.5 Hz, Ar–H), 7.41 (t, 2H,

–

3

J = 8.0 Hz, Ar–H), 7.62 (d, 2H, J = 8.0 Hz, Ar–H), 7.69 (d, 2H, J = 7.5

Hz, Ar–H), 9.54 (s, 1H, NH), 10.23 (s, 1H, NH). ¹³C NMR (125 MHz,

DMSO‑d₆) δ 55.5, 105.3, 110.7, 111.9, 113.0, 115.4, 119.8, 122.4,

123.9, 125.0, 126.3, 129.9, 133.4, 138.6, 139.3. Elemental Analysis:

Calcd: C, 67.65; H, 5.30; N, 21.04. Found: C, 68.02; H, 5.63; N, 21.38.

MS m/z (%): 266 (12.4, M⁺).

2.2.2. Synthesis of 2-(methylthio)-5,6-(un) substituted-1H-benzo[d]

imidazoles (7–9)

(0.01 mol) of compounds 4-6, was stirred with methyl iodide (10

ml), anhydrous potassium carbonate (1 g) in 50 ml acetone for 24 h. The

resultant solution was then evaporated under vacuum and the obtained

residue was crystallized from aqueous ethanol to obtain the mentioned

derivatives in reasonable yields.

2.2.4.2. (Z)-2-(2-(2-chlorobenzylidene) hydrazinyl)-1H-benzo[d]imidazo

le (18). Yield 57.3%, ¹H NMR (500 MHz, DMSO‑d₆) δ 6.73 (s, 1H,

2.2.2.1. 2-(Methylthio)-1H-benzo[d]imidazole (7). Yield 63.4%, ¹H NM

R (500 MHz, DMSO‑d₆) δ 1.95 (s, 3H, CH₃), 7.12 (d, 2H, J = 7.5 Hz,

Ar–H), 7.25 (t, 2H, J = 8.5 Hz, Ar–H), 10.42 (s, 1H, NH). Elemental

Analysis: Calcd: C, 58.51; H, 4.91; N, 17.06; S, 19.52. Found: C, 58.25;

H, 4.95; N, 17.26; S, 19.86. MS m/z (%): 164 (8.4, M⁺).

–

N

CH), 7.21–7.25 (m, 4H, Ar–H), 7.62–7.67 (m, 4H, Ar–H), 9.82 (s,

1H, NH), 11.01 (s, 1H, NH). ¹³C NMR (125 MHz, DMSO‑d₆) δ 113.4,

114.7, 116.0, 117.2, 118.9, 122.0, 123.7, 126.4, 129.9, 130.4, 132.6,

136.2, 138.7, 140.9. Elemental Analysis: Calcd: C, 62.11; H, 4.10; N,

20.70. Found: C, 62.17; H, 4.23; N, 20.92. MS m/z (%): 270 (8.0, M⁺).

2.2.2.2. 5,6-Dimethyl-2-(methylthio)-1H-benzo[d]imidazole (8). Yield 5

5.4%, ¹H NMR (500 MHz, DMSO‑d₆) δ 1.67 (s, 6H, CH₃), 1.99 (s, 3H,

CH₃), 7.59 (s, 2H, Ar–H), 9.95 (s, 1H, NH). Elemental Analysis: Calcd: C,

62.47; H, 6.29; N, 14.57; S, 16.67. Found: C, 62.53; H, 6.64; N, 14.32; S,

16.82. MS m/z (%): 192 (17.0, M⁺).

2.2.4.3. (Z)-2-(2-(4-chlorobenzylidene) hydrazinyl)-1H-benzo[d]imidazo

le (19). Yield 64.9%, ¹H NMR (500 MHz, DMSO‑d₆) δ 6.26 (s, 1H,

–

N

CH), 7.27 (d, 2H, J = 8.5 Hz, Ar–H), 7.31 (d, 2H, J = 8.0 Hz, Ar–H),

–

7.35–7.39 (m, 4H, Ar–H), 9.54 (s, 1H, NH), 11.22 (s, 1H, NH). ¹³C NMR

(125 MHz, DMSO‑d₆) δ 111.0, 114.6, 114.9, 115.1, 116.7, 118.3, 120.6,

122.7, 123.0, 124.5, 127.7, 129.6, 130.4, 138.1. Elemental Analysis:

Calcd: C, 62.11; H, 4.10; N, 20.70. Found: C, 62.21; H, 4.27; N, 21.0. MS

m/z (%): 270 (10.2, M⁺).

2.2.2.3. 5,6-Dichloro-2-(methylthio)-1H-benzo[d]imidazole (9). Yield 5

9.4%, ¹H NMR (500 MHz, DMSO‑d₆) δ 1.95 (s, 3H, CH₃), 8.21 (s, 2H,

Ar–H), 10.01 (s, 1H, NH). Elemental Analysis: Calcd: C, 41.22; H, 2.59;

3

H.A. Elkot et al.

Chemico-Biological Interactions 344 (2021) 109530

2.2.4.4. (Z)-2-(2-(4-nitrobenzylidene) hydrazinyl)-1H-benzo[d]imidazole

NMR (125 MHz, DMSO‑d₆) δ 115.3, 116.7, 120.2, 122.6, 123.7, 124.1,

125.9, 127.2, 129.4, 131.7, 133.3, 136.7. 138.0. Elemental Analysis:

Calcd: C, 49.51; H, 2.67; N, 16.50. Found: C, 49.91; H, 2.01; N, 16.91.

MS m/z (%): 338 (11.0, M⁺).

(20). Yield 56.2%, ¹H NMR (500 MHz, DMSO‑d₆) δ 6.92 (s, 1H,

–

N

CH), 7.26 (d, 2H, J = 7.5 Hz, Ar–H), 7.38 (t, 2H, J = 8.0 Hz, Ar–H),

–

7.40 (d, 2H, J = 7.5 Hz, Ar–H), 7.51 (d, 2H, J = 8.5 Hz, Ar–H), 9.43 (s,

1H, NH), 10.28 (s, 1H, NH). ¹³C NMR (125 MHz, DMSO‑d₆) δ 116.2,

118.7, 119.1, 119.9, 121.0, 122.5, 123.7, 125.8, 127.9, 128.8, 129.4,

133.1, 135.6, 138.0 Elemental Analysis: Calcd: C, 59.78; H, 3.94; N,

24.90. Found: C, 59.99; H, 4.02; N, 25.01. MS m/z (%): 281 (36.2, M⁺).

2.2.4.12. (Z)-5,6-Dichloro-2-(2-(4-nitrobenzylidene)

hydrazinyl)-1H-

benzo[d]imidazole (28). Yield 71.3%, ¹H NMR (500 MHz, DMSO‑d₆) δ

–

6.84 (s, 1H, N CH), 7.24 (s, 2H, Ar–H), 7.50 (m, 4H, Ar–H), 10.24 (s,

–

1H, NH), 10.94 (s, 1H, NH). Elemental Analysis: Calcd: C, 48.02; H,

2.59; N, 20.00 Found: C, 48.51; H, 2.91; N, 20.34. MS m/z (%): 349

(18.7, M⁺).

2.2.4.5. (Z)-2-(2-(4-methoxybenzylidene) hydrazinyl)-5,6-dimethyl-1H-

benzo[d]imidazole (21). Yield 75.8%, ¹H NMR (500 MHz, DMSO‑d₆) δ

–

1.62 (s, 6H, 2CH ), 3.81 (s, 3H, OCH ), 6.82 (s, 1H, N CH), 7.54 (s, 2H,

–

3

Ar–H), 7.71 (d, 2H, J = 8.5 Hz, Ar–H), 7.75 (d, 2H, J = 8.0 Hz, Ar–H),

9.87 (s, 1H, NH), 11.00 (s, 1H, NH). ¹³C NMR (125 MHz, DMSO‑d₆) δ

18.5, 19.2, 55.5, 115.2, 116.4, 118.9, 120.0, 122.4, 124.1, 127.6, 128.1,

130.0, 132.5, 135.5, 137.8, 138.4, 139.2. Elemental Analysis: Calcd: C,

69.37; H, 6.16; N, 19.03. Found: C, 69.42; H, 6.45; N, 19.31. MS m/z

(%): 294 (8.2, M⁺).

2.2.5. General procedure for the synthesis of 3-((5,6-(un) substituted)-1H-

benzo[d]imidazole-2-yl) amino)-2-(substituted phenyl)-1,3-thiazinan-4-

ones (30–41)

Derivatives 17-28 (0.01 mol) were dissolved in 30 ml benzene, then

cyclizing agent mercaptopropanoic acid (1 ml) was added and the

mixture was reﬂuxed overnight. Then, the solvent was evaporated under

reduced pressure and left to obtain ﬁnal crystals of compounds 30-41. A

little amount of sodium carbonate was dissolved in distilled water and

added to the crystals to get rid of the excess mercaptopropanoic acid.

2.2.4.6. (Z)-2-(2-(2-chlorobenzylidene)

hydrazinyl)-5,6-dimethyl-1H-

benzo[d]imidazole (22). Yield 64.1%, ¹H NMR (500 MHz, DMSO‑d₆) δ

–

1.56 (s, 6H, 2CH ), 6.91 (s, 1H, N CH), 7.27 (s, 2H, Ar–H), 7.53 (m, 4H,

–

Ar–H), 10.04 (s³, 1H, NH), 10.99 (s, 1H, NH). ¹³C NMR (125 MHz,

DMSO‑d₆) δ 20.1, 21.3, 115.4, 117.4, 118.1, 120.3, 121.0, 122.7, 125.9,

127.4, 129.4, 131.0, 133.4, 135.6, 137.1, 139.7 Elemental Analysis:

Calcd: C, 64.32; H, 5.06; N, 18.75. Found: C, 64.71; H, 5.19; N, 19.09.

MS m/z (%): 298 (17.4, M⁺).

2.2.5.1. 3-((1H-Benzo[d]imidazole-2-yl) amino)-2-(4-methoxyphenyl)-

1

1,3-thiazinan-4-one (30). Yield 71.5%, H NMR (500 MHz, DMSO‑d₆)

δ 3.70 (s, 3H, OCH₃), 2.73 (t, 2H, J = 1.5 Hz, CH₂), 2.85 (t, 2H, J = 1.5

Hz, CH₂), 4.92 (s, 1H, CH), 7.11 (d, 2H, J = 8.0 Hz, Ar–H), 7.32 (d, 2H, J

= 8.0 Hz, Ar–H), 7.45 (t, 2H, J = 8.0 Hz, Ar–H), 7.53 (d, 2H, J = 7.5 Hz,

Ar–H), 9.82 (s, 1H, NH), 10.10 (s, 1H, NH). Elemental Analysis: Calcd: C,

61.00; H, 5.12; N, 15.81. Found: C, 61.22; H, 5.17; N, 15.91. MS m/z

(%): 354 (19.7, M⁺).

2.2.4.7. (Z)-2-(2-(4-chlorobenzylidene)

hydrazinyl)-5,6-dimethyl-1H-

benzo[d]imidazole (23). Yield 69.9%, ¹H NMR (500 MHz, DMSO‑d₆) δ

–

1.53 (s, 6H, 2CH ), 7.10 (s, 1H, N CH), 7.31 (s, 2H, Ar–H), 7.69 (d, 2H,

–

3

J = 7.5 Hz, Ar–H), 7.74 (d, 2H, J = 8.0 Hz, Ar–H), 10.22 (s, 1H, NH),

11.34 (s, 1H, NH). Elemental Analysis: Calcd: C, 64.32; H, 5.06; N,

18.75. Found: C, 64.95; H, 5.24; N, 19.17. MS m/z (%): 298 (16.4, M⁺).

2.2.5.2. 3-((1H-Benzo[d]imidazole-2-yl) amino)-2-(2-chlorophenyl)-1,3-

thiazinan-4-one (31). Yield 66.4%, ¹H NMR (500 MHz, DMSO‑d₆) δ

2.77 (t, 2H, J = 2.5 Hz, CH₂) 2.93 (t, 2H, J = 2.0 Hz, CH₂), 4.99 (s, 1H,

CH), 7.31–7.38 (m, 4H, Ar–H), 7.52–7.60 (m, 4H, Ar–H), 8.57 (s, 1H,

NH), 9.93 (s, 1H, NH). ¹³C NMR (125 MHz, DMSO‑d₆) δ 25.4, 28.7, 49.3,

114.8, 115.1, 117.3, 119.0, 120.2, 122.9, 124.7, 126.0, 128.6, 131.0,

133.4, 145.3, 146.3, 159.5. Elemental Analysis: Calcd: C, 56.90; H, 4.21;

N, 15.61. Found: C, 57.12; H, 4.43; N, 15.94. MS m/z (%): 358 (16.3,

M⁺).

2.2.4.8. (Z)-5,6-dimethyl-2-(2-(4-nitrobenzylidene) hydrazinyl)-1H-benzo

[d]imidazole (24). Yield 77.3%, ¹H NMR (500 MHz, DMSO‑d₆) δ 1.49 (s,

–

6H, 2CH ), 6.86 (s, 1H, N CH), 7.37 (s, 2H, Ar–H), 7.59 (m, 4H, Ar–H),

–

3

9.99 (s, 1H, NH), 11.01 (s, 1H, NH). ¹³C NMR (125 MHz, DMSO‑d₆) δ

19.8, 21.3, 115.7, 117.9, 118.0, 119.9, 121.4, 122.5, 123.6, 125.0,

127.0, 127.9, 129.6, 133.6, 135.4, 137.4. MS m/z (%): 309 (21.0, M⁺).

2.2.5.3. 3-((1H-Benzo[d]imidazole-2-yl) amino)-2-(4-chlorophenyl)-1,3-

thiazinan-4-one (32). Yield 73.1%, ¹H NMR (500 MHz, DMSO‑d₆) δ

2.61 (t, 2H, J = 2.0 Hz, CH₂), 2.80 (t, 2H, J = 2.5 Hz, CH₂), 5.01 (s, 1H,

CH), 7.00 (d, 2H, J = 8.5 Hz, Ar–H), 7.38 (d, 2H, J = 8.0 Hz, Ar–H),

7.49–7.53 (m, 4H, Ar–H), 9.24 (s, 1H, NH), 9.84 (s, 1H, NH). ¹³C NMR

(125 MHz, DMSO‑d₆) δ 27.3, 27.9, 42.5, 110.5, 113.7, 117.2, 121.0,

122.6, 124.6, 127.1, 130.0, 133.8, 135.4, 141.1, 145.3, 157.2.

Elemental Analysis: Calcd: C, 56.90; H, 4.21; N, 15.61. Found: C, 57.03;

H, 4.53; N, 15.81. MS m/z (%): 358 (7.5, M⁺).

2.2.4.9. (Z)-5,6-Dichloro-2-(2-(4-methoxybenzylidene) hydrazinyl)-1H-

benzo[d]imidazole (25). Yield 65.3%, ¹H NMR (500 MHz, DMSO‑d₆) δ

–

3.71 (s, 3H, OCH ), 6.40 (s, 1H, N CH), 7.13 (s, 1H, Ar–H), 7.26 (s, 1H,

–

3

Ar–H), 7.45 (d, 2H, J = 7.9 Hz, Ar–H), 7.52 (d, 2H, J = 8.0 Hz, Ar–H),

9.91 (s, 1H, NH), 10.00 (s, 1H, NH). ¹³C NMR (125 MHz, DMSO‑d₆) δ

55.5, 104.5, 105.6, 110.1, 111.6, 112.2, 114.8, 117.3, 120.4, 122.6,

123.7, 128.4, 130.6, 133.0. Elemental Analysis: Calcd: C, 53.75; H, 3.61;

N, 16.72. Found: C, 54.01; H, 3.82; N, 16.99. MS m/z (%): 334 (17.4,

M⁺).

2.2.5.4. 3-((1H-benzo[d]imidazole-2-yl) amino)-2-(4-nitrophenyl)-1,3-

thiazinan-4-one (33). Yield 75.4%, ¹H NMR (500 MHz, DMSO‑d₆) δ

2.86 (t, 2H, J = 2.6 Hz, CH₂), 2.89 (t, 2H, J = 2.5 Hz, CH₂), 4.97 (s, 1H,

CH), 7.31–7.35 (m, 4H, Ar–H), 7.43–7.48 (m, 4H, Ar–H), 9.02 (s, 1H,

NH), 9.37 (s, 1H, NH). Elemental Analysis: Calcd: C, 55.28; H, 4.09; N,

18.96. Found: C, 55.73; H, 4.28; N, 19.34.

2.2.4.10. (Z)-5,6-Dichloro-2-(2-(2-chlorobenzylidene) hydrazinyl)-1H-

benzo[d]imidazole (26). Yield 52.3%, ¹H NMR (500 MHz, DMSO‑d₆) δ

–

6.35 (s, 1H, N CH), 7.13 (s, 1H, Ar–H), 7.27 (s, 1H, Ar–H), 7.37–7.42

–

(m, 4H, Ar–H), 9.46 (s, 1H, NH), 10.54 (s, 1H, NH). ¹³C NMR (125 MHz,

DMSO‑d₆) δ 115.2, 116.4, 120.9, 122.6, 123.0, 124.6, 127.0, 129.3,

131.0, 132.7, 133.9, 136.1, 137.5, 139.0. Elemental Analysis: Calcd: C,

49.51; H, 2.67; N, 16.50. Found: C, 49.82; H, 2.94; N, 16.73. MS m/z

(%): 338 (24.6, M⁺).

2.2.5.5. 3-((5,6-Dimethyl-1H-benzo[d]imidazole-2-yl)

amino)-2-(4-

methoxyphenyl)-1,3-thiazinan-4-one (34). Yield 76.2%, ¹H NMR (500

MHz, DMSO‑d₆) δ 1.51 (s, 3H, CH₃), 1.54 (s, 3H, CH₃), 2.70 (t, 2H, J =

1.0 Hz, CH₂), 2.83 (t, 2H, J = 1.5 Hz, CH₂), 3.75 (s, 3H, OCH₃), 5.11 (s,

1H, CH), 7.24 (s, 2H, Ar–H), 7.36 (d, 2H, J = 8.0 Hz, Ar–H), 7.51 (d, 2H,

J = 8.0 Hz, Ar–H), 8.57 (s, 1H, NH), 10.02 (s, 1H, NH). MS m/z (%): 382

(25.3, M⁺).

2.2.4.11. (Z)-5,6-Dichloro-2-(2-(4-chlorobenzylidene) hydrazinyl)-1H-

benzo[d]imidazole (27). Yield 64.3%, ¹H NMR (500 MHz, DMSO‑d₆) δ

–

7.01 (s, 1H, N CH), 7.39 (s, 2H, Ar–H), 7.45 (d, 2H, J = 8.0 Hz, Ar–H),

–

7.56 (d, 2H, J = 8.0 Hz, Ar–H), 9.88 (s, 1H, NH), 11.06 (s, 1H, NH). ¹³

C

4

H.A. Elkot et al.

Chemico-Biological Interactions 344 (2021) 109530

2.2.5.6. 2-(2-Chlorophenyl)-3-((5,6-dimethyl-1H-benzo[d]imidazole-2-

2.3. Screening of in vitro antitumor activity

1

yl) amino)-1,3-thiazinan-4-one (35). Yield 59.1%, H NMR (500 MHz,

DMSO‑d₆) δ 1.63 (s, 3H, CH₃), 1.71 (s, 3H, CH₃), 2.64 (t, 2H, J = 2.0 Hz,

CH₂) 2.87 (t, 2H, J = 2.0 Hz, CH₂), 4.84 (s, 1H, CH), 7.01 (s, 1H, NH),

7.41–7.46 (m, 4H, Ar–H), 7.53 (s, 1H, Ar–H), 7.61 (s, 1H, Ar–H), 9.97 (s,

1H, NH). ¹³C NMR (125 MHz, DMSO‑d₆) δ 19.1, 20.3, 24.7, 29.0, 47.1,

113.9, 115.2, 117.0, 120.4, 122.4, 123.6, 130.8, 132.6, 158.1.

Elemental Analysis: Calcd: C, 58.98; H, 4.95; N, 14.48. Found: C, 29.14;

H, 5.21; N, 14.92. MS m/z (%): 386 (21.0, M⁺).

The synthesized compounds were screened for their antitumor ac-

tivity versus different cancer cell lines; hepatocellular carcinoma

(HEPG-2), breast cancer (MCF-7), and prostate cancer (PC-3). The cell

lines were acquired from ATCC via Holding Company for Biological

Products and Vaccines (VACSERA), Cairo, Egypt. To serve as a standard

anticancer drug, doxorubicin (DOX) was used at serial concentrations

ranging from 1.5 to 100

μM (Positive control). The cytotoxic effect was

determined using the MTT colorimetric assay. In a medium of RPMI-

1640 with 100 U/ml penicillin, 100

μg/ml streptomycin, and 10%

2.2.5.7. 2-(4-Chlorophenyl)-3-((5,6-dimethyl-1H-benzo[d]imidazole-2-

FBS, cells were cultured at 37 ^◦C in a 5% CO₂incubator. At a density of

10⁴cells/well, the cell lines were seeded in a 96-well plate. After in-

cubation, cell lines were treated with serial concentrations of the syn-

1

yl) amino)-1,3-thiazinan-4-one (36). Yield 76.3%, H NMR (500 MHz,

DMSO‑d₆) δ 1.68 (s, 3H, CH₃), 1.71 (s, 3H, CH₃), 2.69 (t, 2H, J = 2.0 Hz,

CH₂), 2.82 (t, 2H, J = 2.5 Hz, CH₂), 4.81 (s, 1H, CH), 7.24 (d, 2H, J = 8.0

Hz, Ar–H), 7.29 (s, 1H, Ar–H), 7.34 (d, 2H, J = 8.0 Hz, Ar–H), 7.36 (s,

1H, Ar–H), 9.61 (s, 1H, NH), 9.93 (s, 1H, NH). Elemental Analysis:

Calcd: C, 58.98; H, 4.95; N, 14.48. Found: C, 58.82; H, 5.04; N, 14.73.

MS m/z (%): 386 (23.1, M⁺).

thesized compounds that ranged from 1.5 to 100 μM for 24 h. The

synthesized compounds were dissolved in DMSO. At the end of the in-

cubation period, cells were subjected to the MTT cell viability assay.

Brieﬂy, cells were washed with phosphate buffer saline (PBS), treated

with 20

Then, DMSO (100

μ

l of MTT solution (5 mg/ml), and incubated for 4 h at 37 ^◦C.

μ

l) was added to each well to dissolve the formed

2.2.5.8. 3-((5,6-dimethyl-1H-benzo[d]imidazole-2-yl)

amino)-2-(4-

nitrophenyl)-1,3-thiazinan-4-one (37). Yield 73.1%, ¹H NMR (500

MHz, DMSO‑d₆) δ 1.63 (s, 6H, 2CH₃), 2.81 (t, 2H, J = 1.5 Hz, CH₂), 2.92

(t, 2H, J = 2.5 Hz, CH₂), 5.11 (s, 1H, CH), 7.16–7.20 (m, 4H, Ar–H),

7.49–7.53 (m, 4H, Ar–H), 8.93 (s, 1H, NH), 10.21 (s, 1H, NH). ¹³C NMR

(125 MHz, DMSO‑d₆) δ 15.1, 17.3, 28.4, 30.2, 46.1, 111.5, 113.7, 116.0,

118.2, 121.4, 125.6, 128.0, 128.6, 131.9, 134.2, 139.9, 140.0, 147.2,

157.9. Elemental Analysis: Calcd: C, 57.42; H, 4.82; N, 17.62. Found: C,

57.66; H, 5.13; N, 17.94. MS m/z (%): 397 (8.6, M⁺).

purple formazan crystals. The optical densities of wells were determined

at 570 nm using a microplate reader (Dynatech, Melville, NY, USA).

Experiments were done in triplicates and the viability of non-treated

cells (DMSO control) was used to calculate the mean relative viability

percentage. Furthermore, the concentration required to kill 50% of cells

relative to the control cells was designated as the half-maximal inhibi-

tory concentration (IC₅₀) and it was determined using GraphPad Prism

6.0 (GraphPad Software, USA). Statistical Analysis was done using One-

way ANOVA with Tukey post-hock test. Signiﬁcance was set at P < 0.05.

By taking a closer look at the results obtained, it was revealed that

the target derivatives (30-41) exerted promising activity against the

tested cell lines. Among this series, Compounds 30 and 31 were found to

be the most active derivative. Hence, these two derivatives were sub-

jected to molecular modeling to investigate their potential interaction

with the target protein. Based on the outcomes of the antitumor activity

and docking studies, Compound 30 (Drug) had been selected to undergo

further evaluations. The MTT cell viability assay against normal human

liver cells (THLE-2, VACSERA, Egypt) was performed as described above

after 24 h and 72 h incubation to test the effect of Compound 30 against

the normal cells. Also, Compound 30 was loaded in different formula-

tions of PLGA-based NPs. The physicochemical evaluation of NPs, which

was mentioned later in this study, had directed us to select NP₄as an

optimized formula for further evaluation. Drug and equivalent amounts

2.2.5.9. 3-((5,6-Dichloro-1H-benzo[d]imidazole-2-yl)

amino)-2-(4-

methoxyphenyl)-1,3-thiazinan-4-one (38). Yield 86.4%, ¹H NMR (500

MHz, DMSO‑d₆) δ 2.75 (t, 2H, J = 2.0 Hz, CH₂), 2.82 (t, 2H, J = 1.5 Hz,

CH₂), 3.72 (s, 3H, OCH₃), 4.68 (s, 1H, CH), 7.24 (d, 2H, J = 7.5 Hz,

Ar–H), 7.31 (s, 2H, Ar–H), 7.42 (d, 2H, J = 8.0 Hz, Ar–H), 8.24 (s, 1H,

NH), 9.50 (s, 1H, NH). ¹³C NMR (125 MHz, DMSO‑d₆) δ 26.9, 28.3, 47.0,

112.4, 116.0, 117.4, 120.9, 121.7, 124.1, 126.2, 127.3, 129.0, 130.4,

132.7, 135.6, 138.1, 146.3, 158.4. MS m/z (%): 422 (12.1, M⁺).

2.2.5.10. 2-(2-Chlorophenyl)-3-((5,6-dichloro-1H-benzo[d]imidazole-2-

1

yl) amino)-1,3-thiazinan-4-one (39). Yield 79.1%, H NMR (500 MHz,

DMSO‑d₆) δ 2.75 (t, 2H, J = 2.0 Hz, CH₂), 2.86 (t, 2H, J = 2.5 Hz, CH₂),

4.92 (s, 1H, CH), 7.31 (m, 4H, Ar–H), 7.35 (s, 1H, Ar–H), 7.39 (s, 1H,

Ar–H), 8.41 (s, 1H, NH), 10.31 (s, 1H, NH). Elemental Analysis: Calcd: C,

47.74; H, 3.06; N, 13.10. Found: C, 47.99; H, 3.27; N, 13.73. MS m/z

(%): 426 (16.2, M⁺).

of NP₄were prepared at the concentration range of 1.5–100 μM. Their

antitumor activities were tested as stated above for 24 h and 72 h of

incubation.

2.2.5.11. 2-(4-Chlorophenyl)-3-((5,6-dichloro-1H-benzo[d]imidazole-2-

2.4. Molecular docking

1

yl) amino)-1,3-thiazinan-4-one (40). Yield 66.4%, H NMR (500 MHz,

DMSO‑d₆) δ 2.76–2.79 (m, 4H, 2CH₂), 4.80 (s, 1H, CH), 7.20–7.25 (m,

4H, Ar–H), 7.49 (s, 2H, Ar–H), 10.05 (s, 1H, NH), 10.47 (s, 1H, NH). ¹³C

NMR (125 MHz, DMSO‑d₆) δ 27.4, 29.3, 47.0, 113.7, 114.2, 116.7,

118.0, 119.4, 123.3, 127.2, 129.0, 131.5, 134.8, 136.2, 138.6, 139.2,

159.4. Elemental Analysis: Calcd: C, 47.74; H, 3.06; N, 13.10. Found: C,

47.93; H, 3.24; N, 13.52. MS m/z (%): 426 (10.2, M⁺).

This section of our study had aimed to perform docking and inves-

tigation of the prospective mechanism of action of some of the promising

newly synthesized compounds. Three-dimensional structures of some

substituted amide benzimidazole derivatives to represent the best EZH2

inhibitors, in their neutral forms, were constructed using Molecular

Operating Environment software (MOE of Chemical Computing Group

Inc.). A reported reference molecule with a benzimidazole nucleus and

selective EZH2 inhibition was used [19]. The lowest energy conformer

of new analogs ‘global-minima’ was docked into the binding pocket of

the EZH2 enzyme which is subunit 3 of PRC2 to suggest the mechanism

of epigenetic regulation as antitumor activity. The required PDB protein

under investigation was obtained from the Protein Data Bank of Broo-

khaven National Laboratory. Hydrogens were ﬁrstly added, followed by

the subjection of the enzyme structure to a reﬁnement protocol. The

constraints on the enzyme were progressively removed and minimized

till the RMSD gradient reached the value of 0.01 kcal/mol Å. Molecular

mechanics force ﬁeld ‘AMBER.’ Was used for energy minimization.

2.2.5.12. 3-((5,6-Dichloro-1H-benzo[d]imidazole-2-yl)

amino)-2-(4-

nitrophenyl)-1,3-thiazinan-4-one (41). Yield 69.5%, ¹H NMR (500

MHz, DMSO‑d₆) δ 2.82–2.90 (m, 4H, 2CH₂), 5.25 (s, 1H, CH), 7.26–7.30

(m, 4H, Ar–H), 7.51–7.57 (m, 4H, Ar–H), 8.93 (s, 1H, NH), 9.64 (s, 1H,

NH). ¹³C NMR (125 MHz, DMSO‑d₆) δ 28.7, 30.7, 45.1, 114.9, 116.1,

118.0, 119.9, 122.4, 125.8, 127.3, 129.1, 130.0, 135.7, 141.6, 158.2.

Elemental Analysis: Calcd: C, 46.59; H, 2.99; N, 15.98. Found: C, 46.86;

H, 3.12; N, 16.34. MS m/z (%): 437 (16.8, M⁺).

5

H.A. Elkot et al.

Chemico-Biological Interactions 344 (2021) 109530

Energy minimizations (EM) were performed for each benzimidazole

derivative using 1000 steps of steepest descent. After that, the conjugate

gradient minimization to an RMSD energy gradient of 0.01 kcal/mol Å

was acquired. By using a radius of 10.0 Å around MTX, the active site of

the enzyme was distinguished. Binding energy was calculated as the

difference between the complex energy and the individual energies of

the ligand and enzyme. The compounds under docking study were

subjected to a ﬂexible alignment experiment using MOE. The builder

module of MOE was used to construct the structure of the molecules.

Their geometry was enhanced by using the MMFF94 force ﬁeld. The

Lowest energy-aligned conformers were identiﬁed and have been used

in the docking study.

Table 1

Composition and physicochemical properties of the prepared Drug-loaded NPs.

Ingredient % W/V

Formulation code

NP₁

NP₂

NP₃

NP₄

PLGA

1

0.6

0.06

2

1

0.6

0.06

-

Compound 30

0.1

2

0.1

-

PVA

T₂₀

-

2

Item

Z-average

PDI

Evaluation (mean ± SD)

Almost all polymer

separated as coarse

agglomerates and no

formation of NPs

252.2 ± 17.4

0.18 ± 0.05

ꢀ 10.04 ± 3.96

14.9 ± 7.41

1.56 ± 0.15

18.14 ± 8.1

256.2 ± 4.8

0.19 ± 0.03

ꢀ 16.58 ± 4.4

35.41 ± 5.33

4.15 ± 0.43

46.19 ± 6.72

ζ

EE%

LC%

Y%

2.5. Preformulation study

Z-average, Average size; PDI, polydispersity index; ζ, zeta potential, EE%,

encapsulation efﬁciency; LC%, loading capacity; Y%, percent yield.

2.5.1. The solubility of Compound 30

The solubility of Compound 30 was determined in deionized water

(DIW) and in phosphate buffer pH 7.4 containing 20% v/v methanol

(PB_7.4/MeOH₂₀) as solvents. An excess amount of Compound 30 (10 mg)

was transferred to screw-capped tubes and 5 mL of solvent were added.

Then, the tubes were secured and continuously shaken in a reciprocating

water bath (Fisher Scientiﬁc, USA) at 37 ± 0.5 ^◦C for 72 h to attain

equilibrium. Excess undissolved Compound 30 was removed by centri-

fugation at 5000 rpm for 30 min. The supernatant was separated, ﬁltered

pharmacokinetics [21].

2.5.3. Fourier transform infrared spectroscopy (FT-IR)

FT-IR spectroscopy was performed for unprocessed Compound 30,

PLGA, the physical binary mixture, and later the formed polymeric NPs;

NP_3,and NP₄at room temperature. Samples were physically mixed with

a suitable amount of potassium bromide using a mortar and pestle,

compressed using a hydraulic press to a disc, and placed in a diffused

reﬂectance cell (Thermo Fisher Scientiﬁc, Inc., Waltham, MA, USA). The

spectra of samples were scanned over wave number in a range of

through a 0.45 μm millipore ﬁlter, and analyzed spectrophotometrically

at 290 nm (λ_max) after appropriate dilution (UV-VIS Spectro double

beam, Labomed Inc., USA). A pre-determined spectrum of Compound 30

showed that the optimal absorbance peak was at 290 nm. Therefore,

290 nm was used as λ_maxto construct a standard curve that was found to

comply with Beer-Lambert’s Law (R²= 0.9994). A range of known

concentrations was prepared so that the measured absorbance was

maintaining values below 1. Triplicates of each solvent were achieved,

and the measured absorbance was converted into concentration ac-

cording to the following equation:

400–4000 cm^{ꢀ 1}

.

2.5.4. Thermal analysis

Thermal analysis in our study was accomplished by Differential

Scanning Calorimetry (DSC). This technique was carried out to assess

the thermal behavior of unprocessed Compound 30, PLGA, the physical

binary mixture, and later the formed polymeric NPs. Samples (5 mg

each) were weighed in aluminum pans using an empty open aluminum

pan as a reference. The samples were placed in the DSC apparatus

(Perkin-Elmer, model DSC-4, New York, NY) and heated in the range of

0–400 ^◦C at a heating rate of 10 ^◦C/min.

Y = 0.05399X + 0.005854

Where Y is the absorbance at 290 nm at the corresponding concentration

(X).

2.6. Preparation of drug-loaded NPs

2.5.2. Octanol-water partition coefﬁcient and log P of Compound 30

K_o/wand log P values of Compound 30 were determined using the

method reported by Jesus et al. with some modiﬁcations [20]. Water

and n-octanol were co-saturated with each other by mixing equals

amounts of them at 37 ± 0.5 ^◦C for 72 h. Then, the obtained biphasic

mixture was transferred to a separating funnel and left for 48 h to allow

full separation of the two layers. The octanol-saturated water was

pipetted and used to prepare a Compound 30-saturated aqueous solution

with a known concentration (C_Total). After that, equal amounts of the

water-saturated organic phase and Compound 30-saturated aqueous

phase were transferred to screw-capped glass tubes that were continu-

PLGA-based NPs were prepared by nanoprecipitation method as re-

ported by Katiyar et al. with some adjustments [22]. Brieﬂy, PLGA was

dissolved in acetone at two concentrations 1% and 0.6% (Table 1) and to

this Compound 30 was added at a ratio of 1:10 of the dry polymer

weight. Four milliliters of the obtained polymeric organic phase con-

taining Compound 30 was added dropwise to 40 ml of aqueous phase

having stabilizer (PVA or Tween 20) at a concentration of 2% w/v under

magnetic stirring (Hot Plate and Magnetic stirrer, Misung Scientiﬁc Co.,

LTD, Korea). The ﬂow rate of the organic phase was maintained at 0.6

ml/min using a 24-gauge needle attached to a syringe. By completing

the addition of organic phase, the obtained mixture was placed in an ice

bath and probe-sonicated using pulsed mode (on 2 s, off 1 s) for 5 min at

100% amplitude (Sonics Vibra cell, Sonic and Materials, Inc., USA). To

enable the evaporation of acetone and leaving behind polymeric NPs

dispersed in the aqueous phase, the obtained dispersion was stirred

overnight. Before centrifugation, any agglomerates in the dispersion

were removed by ﬁltration through Whatman No. 1 ﬁlter paper, and the

ﬁltrate was separated at 11,000 rpm for 1 h (Benchtop Centrifuge, Sigma

Laborzentrifugen, Germany). Then, NPs pellets were washed by DIW,

collected, and stored at ꢀ 20 ^◦C. NPs were dried by lyophilization under

reduced pressure and at ꢀ 80 ^◦C (Freeze dryer, SIM FD8-8T, SIM Inter-

◦

ously mixed in a reciprocating water bath at 37 ± 0.5 C for 72 h to

attain equilibrium. The mixture of organic and aqueous layers was then

left until complete separation. The concentration of Compound 30 in the

aqueous phase was analyzed spectrophotometrically at 290 nm using a

pre-constructed calibration curve (C_Aqueous). K_o/wand log P were

determined using the following equations [20]:

C_OctanolC_Totalꢀ C_Aqueous

K_o/w

=

C_Aqueous

log P = Log K_o/w

Predictable values of aqueous solubility and partition coefﬁcient of

Compound 30 were also determined using the Swiss ADME web tool

(http://www.swissadme.ch) that provides open access to a group of fast

predictive models for determination of physicochemical properties and

◦

national, USA) and the resultant lyophilizates were stored at 4 C for

further valuation. Plain NPs (NP_P) were also prepared by the same steps

mentioned above except for the Drug.

6

H.A. Elkot et al.

Chemico-Biological Interactions 344 (2021) 109530

Scheme 1. Synthesis of the target compounds 30–41.

2.7. Characterization of nanoparticles

measured spectrophotometrically at 290 nm against the corresponding

supernatant of NP_pas a blank. Plain supernatant was used to preclude

any effect of the background. The encapsulation efﬁciency (EE%), the

loading capacity (LC%), and percent yield (Y%) of the NPs were calcu-

lated [23].

2.7.1. Particle size, polydispersity index, and zeta potential

Average size (Z-average), polydispersity index (PDI), and zeta po-

tential (ζ) of NPs were measured. The measurements were performed

after appropriate dilution of aqueous dispersions of the prepared NPs

with DIW at 25 ^◦C (Zetasizer Nanoseries, Malvern Instruments Limited,

UK). The measurements were repeated thrice, and the results were

calculated as mean ± SD. Dynamic light scattering (DLS) and laser

Doppler microelectrophoresis techniques were applied to Z-average and

ζ measurements, respectively.

2.7.4. In vitro release proﬁles and kinetic analysis

Release proﬁles of Compound 30 from Drug-loaded NPs (NP₃and

NP₄) and free Compound 30 were studied via vertical Franz diffusion

cells with some modiﬁcations [24]. To serve as a release medium, 30 ml

of PB_7.4/MeOH₂₀were transferred to each receptor half-cell. Methanol

was added to the release medium to maintain sink conditions [25].

Pre-equilibrated dialysis membrane (Spectra/Por® Dialysis Membrane,

12 KDa, Spectrum Laboratories, Inc., CA, USA) with area 3.8 cm²was

used as a barrier to separate donor and receptor compartments. Then,

NP₃, NP_4,and Free Compound 30 at amounts equivalent to 2 mg of

Compound 30 were suspended in 2 ml of PB_7.4and transferred to the

donor compartments. To avoid evaporation of water, donor compart-

ments were secured with wax foil (Paraﬁlm®_◦M, Bemis Company Inc.,

2.7.2. Morphology study

NPs shape and morphology were determined by transmission elec-

tron microscopy (TEM, JOEL 1010; JEOL Ltd, Tokyo, Japan). A ten-fold

diluted drop of fresh NPs dispersion was positioned on a carbon-coated

copper grid and left for 1 min to allow attachment of some NPs to the

carbon substrate. Then, the extra dispersion was removed, and the grid

was rinsed with DIW twice for a few seconds. After complete drying at

room temperature, TEM photographs were captured directly without

staining.

◦

USA). In a shaking incubator at 37 C ± 0.2 C (GFL, Gesellschaft fur

Labortechnik GmbH, Germany), triplicates of each diffusion cell were

agitated at 100 rpm. Samples of receptor media were withdrawn at a

prearranged schedule and replaced with an equivalent aliquot of fresh

2.7.3. Encapsulation efﬁciency, loading efﬁciency, and percent yield

The percentage of Compound 30 encapsulation efﬁciency was esti-

mated indirectly by determining the amount of free Compound 30 (non-

encapsulated drug) in the supernatant of Drug-loaded NPs dispersion

after centrifugation at 11,000 rpm for 1 h. Free Compound 30 was

medium. The samples were passed through a 0.45 μm ﬁlter, appropri-

ately diluted and their UV absorbances were measured at 290 nm.

Concentrations of the Drug in the release media were determined at each

time interval based on the standard curve that early constructed as

7

H.A. Elkot et al.

Chemico-Biological Interactions 344 (2021) 109530

Table 2

Table 3

Physicochemical characters of the synthesized compounds 30-41.

Percent inhibition and IC₅₀

(

μ

M) of the synthesized compounds 30-41 against

three human cancer cell lines.

Compound

no.

Compound no.

R¹

R²

T_M

Molecular Formula

% Inhibition*

IC₅₀(μM)

30

31

32

33

34

35

36

37

38

39

40

41

H

4-OCH₃

2-Cl

245–6

169–71

142–6

94–6

C₁₈H₁₈N₄O₂S

C₁₇H₁₅ClN₄OS

H

HEPG-

2

MCF-

PC-

3

HEPG-2

MCF-7

PC-3

H

4-Cl

C₁₇H₁₅ClN₄OS

7

H

4-NO₂

4-OCH₃

2-Cl

C_{17 15}

C_{20 22}

C_{19 19}

C_{18 16}

H

N₅O₃S

30

31

32

33

34

35

36

37

38

39

40

41

DOX

64.0

59.9

35.0

21.8

42.6

36.5

29.8

51.7

48.6

33.6

39.8

49.0

72.7

50.8

36.7

63.5

36.1

18.6

40.3

35.2

24.7

50.3

48.2

34.2

24.6

48.3

62.1

8.95

0.06

13.57

1.4

16.64

0.4

27.04

0.2

CH₃

Cl

146–50

155–9

166–9

123–7

181–6

140–6

169–73

102–8

H

N₄O₂S

H

ClN₄OS

N₅O₃S

48.4

33.8

24.9

41.7

37.0

20.4

47.2

47.3

31.5

33.0

51.3

74.1

20.32

1.8

9.41

0.8

4-Cl

H

4-NO₂

4-OCH₃

2-Cl

H

53.29 ±

2.3

74.32 ±

1.9

66.94 ±

1.1

H

Cl₂N₄O₂S

Cl

C₁₇H₁₃Cl₃N₄OS

55.81 ±

3.8

47.64 ±

3.5

64.57 ±

4.1

Cl

4-Cl

C

₁₇H₁₃Cl₃N₄OS

Cl

4-NO₂

C_{17 13}Cl₂N₅O₃S

H

68.27

2.6

59.71

3.8

66.29

0.3

T_M; melting point in ^◦C.

79.27 ±

0.9

91.54 ±

1.1

72.68 ±

1.8

mentioned under the solubility section. The cumulative released con-

centration of the Drug at each time interval was calculated. Using the

following mathematical models: zero-order; ﬁrst-order, Higuchi equa-

tion [26], and Korsmeyer-Peppas equation [27], the acquired release

data were kinetically analyzed. The principal model is the one with the

maximum correlation coefﬁcient (r²) [28]. According to the acquired

results, NP₄was designated for the subsequent investigations.

64.81 ±

3.9

82.47 ±

3.1

72.47 ±

2.2

72.01

2.9

93.48

1.9

85.34

3.3

68.65

1.7

76.76

3.3

71.75

2.8

55.39 ±

4.1

86.47 ±

3.0

63.38 ±

4.5

70.94 ±

2.7

84.36 ±

4.8

75.72 ±

3.8

2.8. Protein analysis of EZH2 and EpCAM in HEPG-2

37.64

2.2

42.84

1.0

40.67

5.1

HEPG-2 cells were cultured in a 6-well plate at a cell density of 10⁵

cell/cm²using DMEM-high glucose with 10% FBS, 100 U/ml penicillin,

4.50

0.2

4.17

0.2

8.87

0.6

and 100 μ

g/ml streptomycin at 37 ^◦C in a 5% CO₂incubator. When cells

HEPG-2, Human liver cancer cell line; MCF-7, Human prostate cancer cell line;

PC-3, Breast cancer cell line; DOX, Doxorubicin, *The presented % inhibitions

reached 90% conﬂuency, FBS was decreased to 2% and cell treatment

was conducted. The Drug was dissolved in DMSO and added to cells to

are at 12.5 μM.

reach a ﬁnal concentration of 50

μM and 100 μM to represent Drug (50

μ

M) group and Drug (1000 μM) group, respectively with DMSO ﬁnal

13-16. The cyclization of Schiff bases was conducted through the reac-

tion with thiopropionic acid (29) and yielded the tetrahydrothiazine

derivatives (30-41). All the newly synthesized compounds were sub-

jected to structural elucidation by elemental analysis, NMR technique,

and mass spectroscopy to conﬁrm the structures of the synthesized

compounds as shown in Table 2.

concentration of 0.2% V/V in both groups. To serve as a control to Drug

groups, additional cells were treated with DMSO only at the same con-

centration (DMSO group). On the other hand, NP₄was dispersed in PBS,

sonicated for 10 min, and then added to another cell lines with Drug

equivalent concentration of 50

(NP₄(100

treating cells with amounts of polymer equivalent to those present in

NP₄(50 M) group (Polymer (50 M) group) and NP₄(100 M) group

μM (NP₄(50

μM) group) and 100 μM

μ

M) group). The polymer (PLGA) was used as a control by

3.2. Screening of antitumor activity of the synthesized compounds

μ

(Polymer (100 μM) group). Also, a control of untreated HEPG-2 cells was

Table 3 documented the antitumor activity of the synthesized com-

conducted (Control group).

pounds expressed as IC₅₀and % inhibition at 12.5 μM. The synthesized

After 72 h of incubation, representative phase-contrast images were

taken. Cells in all groups were trypsinized, washed twice with PBS, then

lysed using heat-freeze shock for EZH2 or speciﬁc kit extraction buffer

for EpCAM measurements. Cell lysates were analyzed for EZH2 and

EpCAM protein expression levels using ELISA Kits. Optical density was

measured using a microplate reader (ROBONIK P2000-India). Experi-

ments were done in triplicates and means were compared using one-way

ANOVA with Tukey post-hock test. Statistical analysis was done using

IBM SPSS Statistics for Windows, Version 25.0. Armonk, NY: IBM Corp.

compounds were subjected to screening against HEPG-2, MCF-7, and

PC-3 to investigate their antitumor activity. By taking a closer look at the

results obtained, it was revealed that the target derivatives 30-41

exerted promising activity against the selected cell lines. Among this

series, compounds 30 and 31 are the most active derivatives with %

inhibition values ranged from 36.7% to 64% and from 48.4% to 63.5%,

respectively. At the same time, these two derivatives exerted the lowest

IC₅₀values among all compounds with values in the range of

8.95–27.04

μM and 9.41–20.32 μM, respectively. Some of the com-

pounds at the same series represent moderate % activity against the

tested cell line such as compounds 34, 37, 38, and 41 with values

ranging 40.3%-51.7% and with IC₅₀values ranging from 37.64 to 93.48

3. Results and discussion

3.1. Chemistry results

μ

M. The rest of the compounds are considered inactive with low % in-

hibition value and high IC₅₀values. Hence, Compounds 30 and 31 were

The synthetic strategy for the synthesis of the target compounds is

illustrated in Scheme 1. Starting with the preparation of 1H-benzo[d]

imidazole-2-thiol derivatives 4-6 by the reaction of 1,2-diaminobenzen

analogs 1-3 with carbon disulﬁde in presence of an alcoholic solution

of potassium hydroxide. Compounds 4-6 were then S-alkylated with

methyl iodide in acetone to produce compounds 7-9 that were treated

with hydrazine hydrate to yield the 2-hydrazinyl-1H-benzo[d]imidazole

derivatives 10-12. Schiff’s bases 17-28 were synthesized through the

reaction of compounds 10-12 with a variety of substituted benzaldehyde

subjected to modeling.

3.3. Structure-activity relationship

It was found that the introduction of methoxy and chloro groups

enhanced the antitumor activity giving the best results especially against

HEPG-2 and MCF-7 cell lines with % Inhibition of 64%, 50.8% and

59.9%, 48.4% of compounds 30 and 31, respectively (Tables 2 and 3).

8

H.A. Elkot et al.

Chemico-Biological Interactions 344 (2021) 109530

compounds (30 and 31) with amino acids (similarities are underlined).

3D and 2D binding of Reference drug with pocket showed that Tyr A111

was a key amino acid (Fig. 1A and 1B). Compounds 30 and 31 were

found to exhibit binding poses like those of the Reference drug occu-

pying the binding cavity and, to varying extents. Compound 30 showed

similarity in binding with Tyr A111 and Tyr A661 (2 bonds) which were

all like the reference drug binding. Compound 31 showed similarity in

binding with Tyr A661, Arg B236. Hence, docking results showed po-

tential binding between Compound 30 and the key amino acid (Tyr

A111). Reference drug and Compound 30 alignment is illustrated in

Fig. 1C. Summing up all the above-mentioned ﬁndings, Compound 30

(Drug) was nominated as a promising EZH2 inhibitor to be loaded in

PLGA-based NPs as drug delivery systems.

Table 4

Best results of ligand docking with EZH2 subunit 3 as anti-tumor.

Reference drug

Compound 30

Compound 31

Tyr A661

Tyr A111

Arg B236

Leu A128

Tyr A111

Tyr A661

—

Tyr A661

Arg B236

Asn A130

—

While it was noticed that the introduction of methyl group might result

in moderate activities of some compounds such as 34, 38, and 41 with %

Inhibition ranging from 40.3% to 51.3%. The compounds having the

lowest activity such as 36 and 40 were structurally similar in having

either two chloro groups or one chloro group and one methyl group with

% Inhibition ranging from 20.4% to 39.8%. It was also worth noting that

the mono nitro derivative of the synthesized series 33 was showing weak

antitumor activity with a % Inhibition of 21.8 against the HEPG-2 cell

line. Upon the introduction of the methyl group along with the nitro

group (compound 37), the activity had shifted towards the direction of

increasing the potency of the antitumor activity to % Inhibition of 51.7

against the same cell line (HEPG-2).

3.5. Preformulation study

3.5.1. T_M, solubility, and LogP of Compound 30

The determined value of T_Mwas found to be about 246 ^◦C. Practi-

cally determined solubilities of Compound 30 were 1.02 ± 0.06 mg/ml

(LogS = ꢀ 2.54, 2.86 mM) and 1.86 ± 0.06 mg/ml (LogS = ꢀ 2.28, 5.22

mM) in DIW and PB_7.4/MeOH₂₀, respectively. While the predicted

aqueous solubility of Compound 30 was found to 0.165 mg/ml (LogS =

ꢀ 3.35, 0.463 mM). According to the USP solubility categorization,

Compound 30 could be listed as slightly soluble (SS) (range of SS is from

1 to 10 mg/ml) [29]. If we rely upon the predicted aqueous solubility,

Compound 30 can be shifted to the very slightly soluble (VSS) category

(0.1–1 mg/ml).

3.4. Molecular docking

Molecular docking permitted to determine the orientation of com-

pounds and binding afﬁnities at the active site of the PRC2. The protein-

ligand complex was studied based on the X-ray crystal structure of PRC2.

To investigate the possible binding mode of interactions, molecular

docking was completed representing antitumor activity into subunit 3 of

the PRC2 protein complex (EZH2). The recently solved X-ray crystal

structure of the PDB ID: 5ls6 was used in our docking study.

For a molecule to dissolve in a solvent, it must be capable to disso-

ciate from its solid crystal lattice by overcoming the intermolecular in-

teractions with other neighboring molecules. Usually, T_Mis used to

determine whether compounds display solid-state-limited solubility or

¨

Table 4 shows the binding of the reference drug and the selected

not [30]. Bergstrom and co-workers stated that compounds with T_M

Fig. 1. Binding between reference drug and pocket 2D (A), 3D (B), reference drug, and Compound 30 alignment (C).

9

H.A. Elkot et al.

Chemico-Biological Interactions 344 (2021) 109530

in water to relax the tight junctions between its molecules with subse-

quent improvement of the solubility. Typically, such components are

cosolvents (e.g., methanol). Such an explanation could be used to

interpret the highly signiﬁcant increase of solubility of Compound 30 in

PB_7.4/MeOH₂₀(student t-test, P < 0.0001). PB_7.4/MeOH₂₀was used later

as release media to impart sink condition. To sum up, Compound 30 can

be considered as a poorly water-soluble compound.

Practically determined values of K_o/wand LogP were 41.31 ± 3.1 and

1.62 ± 0.03, respectively. Comparable predicted values of K_o/wand LogP

were found values (50.12 and 1.7, respectively). Compounds could be

classiﬁed according to their partition coefﬁcients as the following; LogP

< 1 (low lipophilicity), LogP in the range of 1–4 (intermediate lip-

ophilicity) and LogP > 4 (high lipophilicity) [30]. Hence, Compound 30

could be considered as an intermediate lipophilic compound. In the

background of pharmacokinetics, LogP can be valuable to predict the

distribution proﬁle of a drug in vivo which in turn has a great impact on

its absorption, distribution, metabolism, and elimination (ADME). LogP

as well helps to decide the most possible route of administration. For

orally absorbed drugs, they must pass ﬁrst across lipid bilayers of the

intestinal epithelium. Consequently, a certain level of lipophilicity is

essential to allow partitioning of the drug to the bilayer. At the same

time, the drug requires some hydrophilicity so that it will partition out

again to the aqueous environment of systemic circulation [32].

The modiﬁed general solubility equation (GSE) also displayed the

effect of LogP and T_Mon the solubility of a compound [33].

Fig. 2. FT-IR spectra of pure unprocessed Compound 30 (A), PLGA (B), the

physical binary mixture (C), NP₃(D), and NP₄(E).

higher than 200 ^◦C (T_Mof Compound 30 = 246 ^◦C) have strong crystal

lattice that signiﬁcantly inﬂuences their solubility [31]. Throughout the

dissolution of a compound from its solid crystal lattice, the surrounding

water molecules need to make a cavity for the incorporation of a new

compound molecule within the aqueous environment. To decrease the

energy accompanying cavity formation, some additives can be included

LogS = 0.5 ꢀ 0.01(T_Mꢀ 25) ꢀ LogP

Where LogS is the solubility, T_Mis the melting point and LogP is the

partition coefﬁcient. In that way, it was proven that there is a direct

relationship between the partition coefﬁcient of a compound and its

solubility. The solubility of compounds with LogP < 2 (LogP of Com-

pound 30 = 1.62) is principally dependent on their solid state. It is

believed that the drug development process of such compounds, which

are especially solubility-limited due to their solid states, is going

smoothly and terminated relatively early. Such ﬁndings had encouraged

us to go forward in our drug development steps of Compound 30. Any

strategy that can change the solid crystal form could be beneﬁcial for

increasing the rate of dissolution and attaining enhanced apparent sol-

ubility [34]. As a rule, amorphous solid dispersions can offer high

bioavailability for drugs suffering from low solubility. Very high surface

area dispersions, such as Drug-loaded NPs, are principally well appro-

priate to rapid-onset applications owing to their initial fast release rate

[35]. Furthermore, the observed amorphous nature of NPs could impart

higher concentrations of the dissolved drug than crystalline solubility.

Such a feature might contribute to faster and higher absorption rates

[36].

3.5.2. FT-IR

FT-IR is mainly used to measure the selective absorption of light by

the vibration pattern of speciﬁc chemical bonds. Such observation could

permit assessing any kind of interaction that might occur between drug

and polymer. In the case of interaction, the vibrations of the atoms

involved in this interaction can suffer modiﬁcations in frequency and

intensity [37]. Fig. 2 shows the FT-IR spectra of pure unprocessed

Compound 30 (A), PLGA (B), the physical binary mixture (C), NP₃(D),

and NP₄(E) that were studied to assess the interaction between the

Compound 30 and the PLGA [18].

For the spectrum of Compound 30 (Fig. 2A), stretching of N–H and

aromatic C–H exhibited at 3450 cm^{ꢀ 1}and 3116 cm^{ꢀ 1}, respectively.

Small peaks at 2571 cm^{ꢀ 1}and 2660 cm^{ꢀ 1}represented S–H vibration.

While sharp peaks at 1402 cm^{ꢀ 1}and 1254 cm^{ꢀ 1}displayed C–N

–

stretching. Stretching of C N in the imidazole ring and amide C O in

–

the thiazinanone ring appeared at 1466 cm^{ꢀ 1}and 1703 cm^{ꢀ 1}, respec-

tively. The vibrational peak at 1179 cm^{ꢀ 1}could be assigned to the

methoxy C–O bond. Stretching of C C appeared at 1609 cm^{ꢀ 1}and C–C

Fig. 3. Thermograms of pure unprocessed Compound 30 (A), PLGA (B), the

–

physical binary mixture (C), NP₃(D), and NP₄(E).

10

H.A. Elkot et al.

Chemico-Biological Interactions 344 (2021) 109530

appeared at 1308 cm^{ꢀ 1}and 1357 cm^{ꢀ 1}. Methyl group vibration

exhibited at 2882 cm^{ꢀ 1}and 2981 cm^{ꢀ 1}. Fig. 2B displays several major

(PLGA) and Compound 30 (Drug) are dissolved in an organic phase,

which is further added to an aqueous phase containing a stabilizer.

Physical coating of polymer surface by the stabilizer imparts a hydro-

philic stealth character to stabilize polymeric NPs [40]. Although

chlorinated solvents such as methylene chloride and chloroform have

been comprehensively used to dissolve the PLGA, acetone was used.

Acetone has a low boiling point (56 ^◦C) with relatively low toxicity and

it is completely miscible with water [41]. Upon the organic

phase-dropping, droplets are promptly stabilized and then acetone

started to diffuse out of the organic droplets to the surrounding aqueous

medium due to their mutual miscibility. Such a condition creates

interfacial turbulence followed by precipitation of PLGA that ﬁnally

resulted in the formation of NPs [42].

–

peaks in the FT-IR spectrum of PLGA. The carbonyl (C O) group of the

–

PLGA appears at 1749 cm^{ꢀ 1}. Also, bending vibration of the aliphatic

–CH₃and –CH₂groups appear at 1453 cm^{ꢀ 1}, 1384 cm^{ꢀ 1,}and 1270

cm^{ꢀ 1}, whereas the bands at 1180 cm^{ꢀ 1}and 1085 cm^{ꢀ 1}could be related

to the stretching of the ester C–O–C. Absorption peaks between 2850

cm^{ꢀ 1}and 3000 cm^{ꢀ 1}were assigned to stretching vibrations of aliphatic

C–H. The spectrum of Compound 30 and PLGA physical mixture

(Fig. 2C) shows both the characteristic peaks of Compound 30 and PLGA

that presented above in their spectra without major shifts. The only

decrease in the intensity of the peaks could be observed in the spectrum

of the physical mixture. This indicated that there was no interaction

between Compound 30 and PLGA. Fig. 2D and E shows the FT-IR spectra

of NP₃and NP₄, respectively. The characteristic peaks of Compound 30

at lower intensity could be observed due to its proportion in the

formulation. The above-mentioned results revealed computability be-

tween Compound 30 and PLGA and at the same time indicated the

ability to incorporate Compound 30 in the PLGA polymeric matrix of

NPs.

For the Drug-loaded NPs, Compound 30 was incorporated at a ratio

of 1:10 of the dry polymer weight. Using a similar ratio of 1–10, the

organic phase was dropped into the aqueous phase containing the sta-

bilizer (2% w/v). During the preparation of the Drug-loaded NPs, it was

observed that PLGA of NP₁and NP₂almost had separated from the

preparation medium as coarse agglomerates without the formation of

NPs. Hence, Tween 20 was more efﬁcient than PVA as a stabilizer and it

greatly enhanced the formation of NPs (NP₃and NP₄). Due to its high

hydrophilic-lipophilic balance (HLB) value of 16.7, Tween 20 has

adsorbed on the interface to contribute to further reduction of surface

tension and facilitate dispersion of organic phase in aqueous one [43].

Therefore, Tween 20 could participate in the coverage and cause sta-

bilization of newly formed NPs [44]. As a result, our effort was directed

for further preparation and characterization of Tween 20-stabilized NPs

(NP₃and NP₄).

3.5.3. DSC

Thermal analysis via DSC was accomplished to deﬁne any possible

alteration in the physical features of the components individually and to

evaluate crystalline or amorphous behavior of Drug-loaded NPs [38].

Such study of phase transition could specify the potential changes in the

crystallinity of Compound 30 that might affect both in vitro release and

in vivo cellular uptake [39]. As the crystalline form needs more energy to

separate into the well-organized molecules, low aqueous solubility and

consequently low physiological bioavailability might have arisen.

However, randomly arranged molecules of the amorphous state need

low energy to separate and this situation might be followed by superior

bioavailability [15].

3.6.2. Z-average, PDI, and ζ potential

Z-average of NP₃and NP₄were determined using the DLS technique

and were found to be 252.2 nm and 256.2 nm, respectively (Table 1).

The determined Z-average was found to be within the nano-range of

<1,000 nm. Moreover, it could be observed that increasing polymer

concentration by about 40% (NP₃) had not signiﬁcantly affected the Z-

average. Mainly, particle size signiﬁcantly participates in determining

the biofate of NPs in vivo. Very small NPs (<10 nm) can be quickly

cleared from the general circulation by renal ﬁltration. While NPs with a

diameter higher than 200 nm have the potential to be entrapped in the

reticuloendothelial system (RES)-rich organs such as the liver [45]. NPs

with reduced mean particle size (<300 nm) are more appropriate for

drug release and transport in the bloodstream, extravasation, and

accumulation in the interstitial space of the tumor cell (EPR effect) with

passive targeting [46]. Nevertheless, the transport mechanism of NPs,

the pore cutoff size of numerous tumors has been described to range

between 380 and 780 nm [47]. Among different sized nanocarriers, a

maximum particle size of 400 nm was suggested to be suitable for

extravasation into tumors [48]. Some studies have revealed that NPs

with particle sizes lower than 200 nm are more effective for tumor

extravasation [49].

Fig. 3 shows the thermograms of pure unprocessed Compound 30

(A), PLGA (B), the physical binary mixture (C), NP₃(D), and NP₄(E). A

DSC thermogram of Compound 30 presents two endothermic peaks at

105.34 ^◦C and 250.72 ^◦C that could be related to the liberation of

physically-bound water (evaporation of moisture content) and the

melting point of Compound 30, respectively (Fig. 3A). The previously

determined T_Mwas found to be much closer to that exhibited by thermal

analysis of Compound 30. Such a ﬁnding demonstrated the crystallinity

of Compound 30 in its molecular structure. Another small endothermic

peak appeared at 316 ^◦C that could be correlated to the decomposition of

Compound 30. Fig. 3B shows the thermogram of PLGA with two endo-

thermic peaks at 53.33 ^◦C and 369.47 ^◦C representing polymer its glass

transition and its thermal decomposition, respectively. It was reported

that the glass transition of PLGA happened at the 48–55 ^◦C range, with a

midpoint value at 52 ^◦C. No melting point was detected, due to the

amorphous nature of PLGA [37]. For the binary mixture of Compound

30 and PLGA, it could be observed that the characteristic peaks of PLGA

and decomposition peak of Compound 30 were not affected by their

physical blending (Fig. 3C). In the Drug-loaded NPs thermograms, PLGA

peaks were identiﬁed while endothermic peaks of Compound 30 were

completely disappeared (Fig. 3D and E). This indicated that the incor-

poration of Compound 30 in NPs transformed its physical state from

crystalline to amorphous one. Therefore, it could be proven that Com-

pound 30 was completely loaded in an amorphous state because of the

complete dissolution of its crystals in the PLGA-based polymeric matrix.

Hence, Compound 30 remained dispersed at the molecular level in the

solid dispersion after the formation of NPs [39].

The measured values of PDI were less than 0.2. Where 1 is the

maximum value and 0 is the lowest of PDI; the higher the PDI value

designates NPs to have a variable size range with poor homogeneity and

low stability. Small PDI (<0.4) is appropriate because such a value

suggests narrow size distribution. Henceforth, the obtained PDI values of

prepared Drug-loaded NPs indicated a narrow distribution proﬁle that

could realize the EPR effect, and play an imperative role in tissue

accumulation and renal clearance [18]. To sum up, the obtained values

of Z-average and PDI were in the acceptable range and further evaluation

could be conducted.

An additional factor that plays a vital role in biodistribution and

uptake is the surface charge of the NPs which is usually measured in the

form of zeta potential (ζ potential). Table 1 documented that NPs had

negative ζ potential (ꢀ 10.04 mV and ꢀ 16.58 mV). The negative ζ values

could be attributed to the terminal carboxyl groups of PLGA that lead to

negatively charged particle surfaces [50]. The surface charge shows a

3.6. Characterization of nanoparticles

3.6.1. Preparation of drug-loaded polymeric nanoparticles

Among the numerous approaches used in the preparation of NPs, the

nanoprecipitation technique was selected. In this technique, polymer

11

H.A. Elkot et al.

Chemico-Biological Interactions 344 (2021) 109530

Fig. 4. TEM images of NP₃(A), NP₄(B), and in vitro release proﬁles (C). Release data displayed as mean ± SD; n = 3, ***P < 0.001.

critical role in cellular uptake through a two-step process, binding of NPs

with cell membrane and internalization inside cells. Also, The ζ potential

is a sign of the colloidal suspension stability [51]. The negative ζ po-

tential of NPs could cause repulsion among them which in turn might

result in precluding their aggregation [52]. Even though positively

charged NPs have displayed superior internalization in vitro, they can

easily bind other healthy cells such as endothelial cells, before reaching

the target tumor cells. Thus, it is empirical to keep the NPs as neutral or

negatively charged as possible [53]. Besides, positively charged NPs

have presented a dose-dependent hemolytic activity while negatively

charged ones did not [17]. Surface characteristics also affect the biofate

of NPs in the systemic circulation [54]. Longer circulation half-life and

less clearance were exhibited by neutral and negatively charged NPs

[55].

shapes [56]. The TEM image also veriﬁed that NPs did not exhibit ag-

gregation and at the same time had a narrow particle distribution. By

matching DLS-measured Z-average with a particle size that was observed

by TEM, a higher value was exhibited by the DLS technique. The anal-

ogous ﬁnding was documented in several studies. Such a situation could

be attributed to the hydrodynamic Z-average of NPs rather than the true

physical size of individual particles was determined by the DLS tech-

nique [57]. Hence, we thought that the actual particle size of the pre-

pared NPs might be smaller than 250 nm (Z-average).

3.6.4. Encapsulation efﬁciency, loading efﬁciency, and percent yield

EE%, LC%, and Y% of NP₃and NP₄are listed in Table 1. It could be

noted that loading of Compound 30 in NP₄(35.41%) signiﬁcantly

enhanced its EE% to more than two folds that of NP₃(14.9%). In the

same manner, NP₄exhibited better values of LC% and Y% (4.15% and

46.19%, respectively) than corresponding ones of NP₃(1.56% and

18.14%, respectively). From our ﬁndings, it was evident that increase

PLGA concentration was associated with a reduction of EE%, LC%, and Y

% of Compound 30. Tween 20 is a smaller surfactant molecule, which

effectively stabilizes NPs by the formation of a thin protective outer

layer followed by a decrease of interfacial tension. For NP₃, the thin

3.6.3. Morphology study

The morphology and the structure of NP₃and NP₄had been inves-

tigated by TEM images as shown in Fig. 4A and 4B, respectively. Both

formulations showed spherical and uniform structure nanosized parti-

cles with a smooth surface. Zhang and co-workers reported that

spherical-shaped NPs are more readily to be engulfed by cells than other

12

H.A. Elkot et al.

Chemico-Biological Interactions 344 (2021) 109530

Furthermore, slow degradation of PLGA principally causes further sus-

tained release of the Drug. Hence, the obtained release behavior is likely

to be a biphasic release. The in vitro release after 216 h had resulted in

overall 16% and 13% Drug depletion from NP₃and NP₄, respectively.

However, the burst effect is advantageous to get a rapid onset of a drug

at the action site, as well as the prolonged release is essential to uphold

the drug effects [15]. It could be observed that the percent released of

the Drug from NP₃and NP₄was signiﬁcantly lower than the corre-

sponding value of the Drug alone (Drug suspension) along the release

time (P < 0.001). The PLGA-based polymeric matrix of NPs was thought

to be responsible for the observed rate-controlling manners of NP₃and

NP₄. In tumor therapy, attaining sustained drug release played an extra

important role in improving the curative effect, preventing tumor cells

to be subjected to a massive concentration of a drug and hence pre-

cluding the development of therapy resistance [61]. For further inter-

pretation of our results, in vitro release data were ﬁtted to different

mathematical models to be kinetically analyzed.

Table 5

Kinetic analysis of Drug released from its suspension, NP₃and NP_4.

Correlation coefﬁcient (r²)

Formulation Code

Drug

NP₃

NP₄

Zero

0.7455

0.9856

0.9455

First

0.8081

0.8185

0.9443

Higuchi

0.8776

0.84

0.9265

0.9318

0.9746

Higuchi

0.9557

0.85

First

Higuchi

Release order

K–P

r²

n

0.8534

0.59

Main mechanism

Non-Fickian diffusion

layer might be insufﬁcient to wet the excessive amount of Compound 30

(40% higher than NP₄) which probably led to a decrease encapsulation

capability of Tween 20 [58].

3.6.5. In vitro release proﬁles and kinetics

Studying kinetics of drug release is essential as it has correlated the in

vitro and in vivo drug responses by relating outcomes of pharmacoki-

netics and release patterns. Different models could be applied, the most

well-matched being the one that best ﬁts the release data. Table 5 shows

correlation coefﬁcients (r²) that are used to deﬁne the best ﬁtting model.

It was found that the release of the Drug from NP₃and NP₄was best

ﬁtted for the Higuchi model with r²values of 0.9443 and 0.9746,

respectively. Such a ﬁnding suggested a diffusion-driven release mech-

anism of the Drug from the prepared NPs. To understand the release

mechanism in-depth, release data were ﬁtted to the Korsmeyer-Peppas

exponential model. The diffusional release exponent (n) of NP₃and

NP₄were 0.836 and 0.85, respectively which designated that they

exhibited a non-Fickian or anomalous diffusion (0.5< n < 1). This means

that Drug release from the prepared NPs was not governed by pure

diffusion mechanism but is rather a combination of Drug diffusion

through NPs and erosion of the polymeric matrix. On the other hand, it

was found that the release of the Drug from its suspension followed ﬁrst-

order kinetics which means that the release of pure Compound 30 was

dependent on its concentration in the donor compartment. Based on the

above-mentioned characterization, NP₄was selected to be subjected to

further evaluation and development.

The in vitro release studies were performed using a dialysis mem-

brane that simulates the ultimate performance in the biological ﬂuids.

Phosphate buffer containing 20% (V/V) methanol (PB_7.4/MeOH₂₀) was

used as a release medium to provide sink conditions as well as bypassing

the interfering effect of Compound 30 low solubility [59]. In the sink

phase, a drug concentration has to be kept at less than 10% of its satu-

ration solubility [60]. The effectiveness of the release study design could

be ensured by checking the release of the Drug from its unprocessed

naked form. The release pattern of the Drug from its suspension, NP_3,

and NP₄are shown in Fig. 4C. The free Drug was almost released within

24 h. This could certify that the dialysis membrane could allow passage

of Compound 30 molecules through its pores. As exhibited, both NPs

formulations demonstrate an initial burst release during the ﬁrst 72 h of

incubation (Fig. 4C). This initial burst release observed in our study has

been commonly perceived in many studies of the polymeric NPs [11].

This initial burst could be related to Compound 30 molecules embedded

at the outermost layers of the polymeric matrix and those adsorbed on

the surface of NPs. The initial burst was followed by a sustained-release

pattern for both types of NPs. The sustained release could be attributed

to the fraction of Compound 30 that entrapped within the core of NPs.

Fig. 5. Relative viability of HEPG-2, MCF-7, and PC-3 cell lines after incubation with serial concentrations of Compound 30 and NP₄for 24 h and 72 h. Data

displayed as mean ± SD; n = 3, *P < 0.05, * *P < 0.01, and ***P < 0.001.

13

H.A. Elkot et al.

Chemico-Biological Interactions 344 (2021) 109530

future in vivo studies should be determined before commencing studies.

To sum up, the results indicated that Compound 30 decreased the

viability of the tested cancer cell lines with reasonable IC₅₀values.

Furthermore, Compound 30 showed promising antitumor activity

versus HEPG-2. At the same time, the incorporation of Compound 30 in

polymeric PLGA-based NPs (NP₄) had not retarded its antitumor activ-

ity. HEPG-2 was the most sensitive for either Compound 30 or NP₄.

Hence, further evaluation was directed towards focusing on protein

expression levels of HEPG-2 (HCC).

3.8. EZH2 and EpCAM protein expression

Fig. 6A and B shows the protein expression levels of EZH2 and

EpCAM, respectively. The Drug displayed a potent inhibitory effect on

the EZH2 protein expression and reduced its level by ≈ 1.5 fold at 50 μM

and ≈1.8 fold at 100

Also, NP₄decreased the EZH2 expression by ≈ 1.6 fold at 50

fold at 100 M concentration compared to the respective polymer con-

trols. The EpCAM protein expression in cell lysate decreased by ≈ 2 fold

in Drug (50 M), ≈2.7 fold in Drug (100 M), ≈2 fold in NP₄(50 M),

and ≈2.8 fold in NP₄(100 M) groups. Consistently, statistical analysis

revealed that the Drug at 50 M and 100 M signiﬁcantly reduced

μ

M concentration compared to the DMSO group.

μM and ≈2

μ

protein expression levels of EZH2 and EpCAM in comparison to the

DMSO group (P < 0.001) and Control group (P < 0.001). Similarly, NP₄

at both concentrations showed a signiﬁcant decrease in EZH2 and

EpCAM levels compared to the corresponding ones of polymer groups (P

< 0.001), respectively.

It was observed that the decrease in EZH2 protein expression had

occurred in a dose-dependent manner (Fig. 6A). This ﬁnding was veri-

ﬁed by the noticeable signiﬁcant differences between EZH2 expression

levels of Drug and NP₄groups at 100

50

similar dose-dependent pattern (Fig. 6B). Drug and NP₄groups at 50

demonstrated signiﬁcantly higher EpCAM levels than the corresponding

groups at 100 M (P < 0.05). On the other hand, it was found that

treating the cells with 50

μM and the corresponding ones at

μ

M (P < 0.01). Moreover, EpCAM protein expression levels showed a

μM

μ

M or 100 μM of NP₄decreased the protein

expression levels insigniﬁcantly when compared to the corresponding

Drug groups, (P > 0.05). There was no signiﬁcant change between the

EZH2 expression levels in the Control group compared to either DMSO,

Fig. 6. Protein expression level of EZH2 (ng/ml) and EpCAM (pg/ml) in cell

lysates of different groups. Data displayed as mean ± SD; n = 3, *P < 0.05, **P

Polymer (50 μM), or Polymer (100 μM) groups. Interestingly, DMSO

signiﬁcantly decreased EpCAM levels compared to the Control group (P

< 0.05). Besides, both Polymer groups showed a signiﬁcant decrease in

EpCAM levels compared to either the Control group (P < 0.001) or the

DMSO group (P < 0.01). The ability of DMSO to lessen EpCAM

expression in HEPG-2 could be attributed to its reported epigenetic and

in vitro catalytic activities on DNA methyltransferase 3a [62].

$

< 0.01, ***P < 0.001, ^#P < 0.05 Vs the control group, P < 0.001 Vs the

control group, ^!P < 0.01 Vs the DMSO group.

3.7. In vitro antitumor activity of Compound 30 and NP₄

Fig. 5 shows the in vitro antitumor activity of Compound 30 and NP₄,

respectively expressed as means relative viability of HEPG-2, MCF-7,

and PC-3 cell lines after incubation with different concentrations for 24

h (right panel) and 72 h (left panel). It was found that Compound 30

reduced the cell viability of all tested cell lines. It could be observed that

Fig. 7 illustrates phase-contrast images of HEPG-2 cell line of

different groups after 72 h. The control group (Fig. 7A) exhibited normal

cell morphology with typical viability. The Drug (50 μM) group showed

senescent and dead cells while most HEPG-2 cells treated with 100

Drug were dead with a marked decrease of cell number (Fig. 7C and D,

respectively). Cells of both Polymer groups at 50 M and 100 M, which

were shown in Fig. 7E and F, respectively demonstrated some dead cells.

Treatment of HEPG-2 with NP₄at a concentration of 50 M caused an

M) group incor-

μM

the most sensitive cell line was HEPG-2 with IC₅₀values of 8.95

7.71 M after 24 and 72 h of incubation, respectively (Fig. 5A and B).

The least sensitive cell line after 24 h was PC-3 with IC₅₀= 27.03 M.

M after

μM and

μ

However, MCF-7 was the least sensitive one with IC₅₀= 11.6

μ

obvious decrease in cell viability (Fig. 7G). NP₄(100

μ

72 h of incubation (Fig. 5B). It was found that Drug-loaded NPs (NP₄)

also diminished cell viability of the tested cancer cell lines as shown in

Fig. 5. Similarly, HEPG-2 was the most sensitive cell line with IC₅₀

porating a higher concentration of the Drug offered nearly complete cell

death (Fig. 7H).

Our results could be interpreted considering the below-mentioned

details. Embryonic-stage active genes are known to be silenced in

adulthood by several mechanisms with the chromatin-modifying PRC2

being at the core of the silencing mechanism. Dysfunction of PRC2 leads

to reactivation of developmentally regulated genes and has been asso-

ciated with several types of tumors. EZH2 or enhancer of zeste 2 is a

catalytic subunit of PRC2. EZH2 and its associated PRC2 complex are the

most signiﬁcantly deregulated epigenetic regulators in primary HCC

[63]. The overexpression of EZH2 in cancer cells leads to independent

values 7.29 μM and 6.16 μM after 24 and 72 h of incubation, respec-

tively. On the other hand, the PC-3 cell line exhibited the highest

resistance with IC₅₀values of 22.99 M and 12.49 M after 24 and 72 h,

respectively (Fig. 5C and D). Compound 30 showed much higher IC₅₀

μ

values (81.01 ± 12.93 and 62.63 ± 4.52

μM after 24 h and 72 h;

respectively) against THLE-2 cells compared to HEPG-2 (Fig. SI-1).

These data suggest that Compound 30 might have a selective enhanced

cytotoxic effect against the tumor cells. A safe concentration range for

14

H.A. Elkot et al.

Chemico-Biological Interactions 344 (2021) 109530

Fig. 7. Phase contrast images of HEPG-2 cell line after 72 h of incubation of Control (A), DMSO (B), Drug 50 μM (C), Drug 100 μM (D), Polymer 50 μM (E), Polymer

100 μM (F), NP₄50 μM (G) and NP₄100 μM (H) groups. Scale Bar: 100 μm.

methylation and Wnt/β-catenin signaling activation and ﬁnally

increased EpCAM expression [64]. Depletion of β-catenin from mature

hepatocytes initiated active proliferation to replace hepatocytes with

new ones expressing β-catenin and EpCAM leading to an expansion of

hepatic progenitor cells and tumor development [65].

NP₄might decrease the possibility of metastasis or tumor relapse after

+

chemo/radiotherapy due to their ability to eradicate EpCAM cancer

stem cells. Such ﬁndings indicated that Drug and NP₄had targeted EZH2

and the downstream signaling pathway that leads to a decline of EpCAM

expression as well as HEPG-2 cell viability.

EpCAM expression in HCC is believed to mark cancer cells with

epithelial-mesenchymal transition capability; i.e; cancer stem cells.

Eventually, this leads to increased tumor malignancy and worsens the

prognosis [66]. Based on their EZH2 inhibitory effect, both Drug and

NP₄decreased EpCAM expression in HEPG-2. Additionally, Drug and

In the literature, the scholars had focused on the synthesis of

different EZH2 inhibitors without further processing towards further

pharmaceutical and biological applications [15–18]. Hence, the practi-

cability, applicability, and sustainability of the developed compounds

are thought to be impaired in the ﬁeld of pharmacy. In our study, EZH2

15

H.A. Elkot et al.

Chemico-Biological Interactions 344 (2021) 109530

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tumor activity. Then, our work was extended to energize the docking

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most appropriate compound (Compound 30). The physicochemical

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˜

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Author contribution

Ghada S. Hassan, Sara T. Al-Rashood and Dr. Noha M. Saleh designed

research; Hoda A. Elkot, Ibrahim Ragab, Noha M. Saleh, Mohamed N.

Amin achieved research practical work; Shahenda M. El-Messery per-

formed molecular modeling; Ghada S. Hassan, Shahenda M. El-Messery,

Noha M. Saleh, Mohamed N. Amin analyzed data and wrote the paper.

[18] K. Anwer, M. Mohammad, E. Ezzeldin, F. Fatima, A. Alalaiwe, M. Iqbal,

Preparation of sustained release apremilast-loaded PLGA nanoparticles: in vitro

characterization and in vivo pharmacokinetic study in rats, IJN 14 (2019)

1587–1595, https://doi.org/10.2147/IJN.S195048.

[19] S.K. Knutson, T.J. Wigle, N.M. Warholic, C.J. Sneeringer, C.J. Allain, C.R. Klaus, J.

D. Sacks, A. Raimondi, C.R. Majer, J. Song, M.P. Scott, L. Jin, J.J. Smith, E.

J. Olhava, R. Chesworth, M.P. Moyer, V.M. Richon, R.A. Copeland, H. Keilhack, R.

M. Pollock, K.W. Kuntz, A selective inhibitor of EZH2 blocks H3K27 methylation

and kills mutant lymphoma cells, Nat. Chem. Biol. 8 (2012) 890–896, https://doi.

org/10.1038/nchembio.1084.

Declaration of competing interest

[20] A.R. Jesus, M.R.C. Soromenho, L.R. Raposo, J.M.S.S. Esperança, P.V. Baptista, A.

R. Fernandes, P.M. Reis, Enhancement of water solubility of poorly water-soluble

drugs by new biocompatible N-acetyl amino acid N-alkyl cholinium-based ionic

liquids, Eur. J. Pharm. Biopharm. 137 (2019) 227–232, https://doi.org/10.1016/j.

ejpb.2019.03.004.

The authors declare that they have no known competing ﬁnancial

interests or personal relationships that could have appeared to inﬂuence

the work reported in this paper.

[21] A. Daina, O. Michielin, V. Zoete, SwissADME: a free web tool to evaluate

pharmacokinetics, drug-likeness and medicinal chemistry friendliness of small

molecules, Sci. Rep. 7 (2017) 1–12, https://doi.org/10.1038/srep42717.

[22] S.S. Katiyar, E. Muntimadugu, T.A. Rafeeqi, A.J. Domb, W. Khan, Co-delivery of

rapamycin- and piperine-loaded polymeric nanoparticles for breast cancer

treatment, Drug Deliv. (2015) 1–9, https://doi.org/10.3109/

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.

org/10.1016/j.cbi.2021.109530.

10717544.2015.1039667.

[23] E. Ramadan, T. Borg, G.M. Abdelghani, N.M. Saleh, Transdermal microneedle-

mediated delivery of polymeric lamivudine-loaded nanoparticles, J. Pharmaceut.

Technol. Drug Res. 5 (2016) 1, https://doi.org/10.7243/2050-120X-5-1.

[24] M.M. Silva, R. Calado, J. Marto, A. Bettencourt, A.J. Almeida, L. Gonçalves,

Chitosan nanoparticles as a mucoadhesive drug delivery system for ocular

administration, Mar. Drugs 15 (2017) 370 .

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Design, synthesis, and antitumor activity of PLGA nanoparticles incorporating a discovered benzimidazole derivative as EZH2 inhibitor

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